Protein-Protein Binding Kinetics by Biolayer Interferometry

Santos-López, Jorge; Gómez, Sara; Fernández, Francisco J.; Vega, M. Cristina

doi:10.1007/978-3-031-52193-5_6

Cited by 1 publication

(1 citation statement)

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“…The functional influence of LDLR on HuH7 Lp(a) uptake might have been mediated by direct binding to Lp(a) or by an indirect effect of LDLR perturbation such as an alteration of cellular metabolism or the expression of other genes. To evaluate for the potential of Lp(a) to directly bind the LDL receptor, we used bio-layer interferometry, a label-free method in which binding to a ligand is detected by a change in the refractive index for white light 58 . We immobilized biotinylated recombinant LDLR extracellular domain to streptavidin-coated sensors and incubated with a range of concentrations of Lp(a) or the positive and negative controls LDL and HDL, respectively.…”

Section: Resultsmentioning

confidence: 99%

Functional interrogation of cellular Lp(a) uptake by genome-scale CRISPR screening

Khan,

Cunha,

Raut

et al. 2024

Preprint

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An elevated level of lipoprotein(a), or Lp(a), in the bloodstream has been causally linked to the development of atherosclerotic cardiovascular disease and calcific aortic valve stenosis. Steady state levels of circulating lipoproteins are modulated by their rate of clearance, but the identity of the Lp(a) uptake receptor(s) has been controversial. In this study, we performed a genome-scale CRISPR screen to functionally interrogate all potential Lp(a) uptake regulators in HuH7 cells. Strikingly, the top positive and negative regulators of Lp(a) uptake in our screen were LDLR and MYLIP, encoding the LDL receptor and its ubiquitin ligase IDOL, respectively. We also found a significant correlation for other genes with established roles in LDLR regulation. No other gene products, including those previously proposed as Lp(a) receptors, exhibited a significant effect on Lp(a) uptake in our screen. We validated the functional influence of LDLR expression on HuH7 Lp(a) uptake, confirmed in vitro binding between the LDLR extracellular domain and purified Lp(a), and detected an association between loss-of-function LDLR variants and increased circulating Lp(a) levels in the UK Biobank cohort. Together, our findings support a central role for the LDL receptor in mediating Lp(a) uptake by hepatocytes.

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Section: Resultsmentioning

confidence: 99%